Method for measuring sialic acid in immunoglobulin G and immunoglobulin G anti-double-stranded DNA antibodies

Abstract

A method for measuring the amount of sialic acid in immunoglobulin G and immunoglobulin G anti-ds DNA antibodies is disclosed. The method for measuring the amount of sialic acid in immunoglobulin G in the present invention uses culture fluid, blood, plasma, or serum to directly measure the amount of sialic acid in immunoglobulin G. Also, using a mouse monoclonal antibody immunoglobulin G as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml in phosphate buffered saline (PBS), produces good results. The method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies has never been done and the present invention produces good results as well.

What is claimed is: 
     
       1. A method for measuring the absolute amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies comprises steps of:
 Step 1: in an enzyme immunoassay petridish, adding 150 μl of 0.5 mg/ml of protamine chloride in each grid and placing said petridish at room temperature for 2 hours; 
 Step 2: washing (three times) with PBS (pH=7.2) and adding 100 μl of 50 μg/ml calf thymus double-stranded DNA overnight at 4° C.; 
 Step 3: preparing oxidized bovine serum albumin, wherein the bovine serum albumin is dissolved in 20 mM potassium periodate (PBS, pH=7.2) and 50 mM sodium acetate (final pH=4.0 of the mixture of sodium periodate and sodium acetate) at 4° C. (for 30 minutes) to obtain a mixture, and the mixture is dialyzed with tris-buffered saline (pH=7.4), and adding (0.1% by volume) of polyoxyethylene (20) sorbitan monolaurate solution to make 1% oxidized bovine serum albumin; 
 Step 4: using PBS-0.5% Tween 20 as a flushing fluid to flush the enzyme immunoassay petridish (four times), adding 300 μl of 1% of oxidation bovine serum albumin into each grid, and placing said petridish at room temperature for 2 hours in order to block non-specific adhesion; 
 Step 5: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding 100 μl of immunoglobulin G isolated from protein G, and placing said petridish at room temperature for two hours; 
 Step 6: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, and adding diluted (500 times) horseradish peroxidase-linked  sambucus nigra  agglutinin lectin (SNA) 100 μl to each grid; 
 Step 7: washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding mixture of tetramethyl benzidine solution 50 μl and peroxide hydrogen solution 50 μl at room temperature for 5 minutes, and adding 100 μl of 0.5N H 2 SO 4  into each grid to prevent the reaction; and 
 Step 8: measuring the absorption value at 450 nm in each grid in the enzyme immunoassay analyzer, wherein a mouse monoclonal antibody immunoglobulin G anti-double-stranded DNA is used as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml and placed at room temperature for two hours. 
 
     
     
       2. The method for measuring the amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies of  claim 1 , wherein said enzyme immunoassay petridish has ninety-six (96) grids.